AlanFung:LabNotes/Probe/2014-1-7

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Revision as of 00:32, 8 January 2014 by >Alan6017518 (→‎Nanodrop)
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GP1 V6 Padlock Probe Production

Overview

  • Expansion PCR
  • Production PCR
  • Exonuclease digestion to generate SS-DNA
  • Removal of amplification adapters
  • Probe purification by PAGE size-selection

Expansion PCR

  • Probe concentration from CustomArray 32.71ng/ul volume 80uL Amount 2.62ug
  • Probe size 146bp

Convert ng/ul to nM

  • Molar Mass of ssDNA probe: 146bp * (325Da/bp) = 146bp * (325 g/mol/bp) = 47450 g/mol
  • (32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
  • I will use 100nM final concentration for the expansion PCR

GP1_V6 Expansion PCR

Components 1x rxn (ul) Final Conc
GP-1 (689nM) 14.51 100nM
KAPA SYBR FAST Master Mix (2X) 50 1X
100uM pAP1V61U 0.4 400nM
100uM AP2V6 0.4 400nM
H2O 34.69  
Total 100  

Nanodrop

  • 10.7ng/ul * 50ul = 535ng
  • 120.44nM

Dilute probes to 10nM

Production PCR (8 wells)

Components 1x rxn (ul) 8x rxn (ul)
10nM 1st round amplicon 0.4 3.2
KAPA SYBR FAST MM (2X) 50 400
100uM AP1V41U 0.4 3.2
100uM AP2V$ 0.4 3.2
H2O 48.8 390.4
Total 100 800